Mast cells play a critical role in IgE-dependent allergic hypersensitivity and the host defense against certain parasites. Cross-linking of the high-affinity IgE receptor (FcepsilonRI) with IgE and multivalent allergen elicits mast cell activation, culminating in the release of a panel of proinflammatory mediators. PKC plays critical roles in mast cell activation. Among various PKC isoforms that are activated by FcepsilonRI cross-linking, PKCaI activity was shown to be specifically regulated by protein-tyrosine kinases (PTKs), Lyn and Syk, in a Btk-dependent manner. We have demonstrated that C-terminal tyrosine residues, Tyr-662 and Tyr-658 of PKCbetaI and PKCalpha, respectively, are phosphorylated by Syk at the plasma membrane upon FcnRI stimulation. This phosphorylation creates the binding site for the Src homology 2 (SH2) domain of Grb-2, an adaptor protein. Recruitment of Grb-2/Sos complexes to the vicinity of Ras contributes to activation of the Ras/ERK pathway. Akt was also shown to be activated and involved in cytokine production upon FcepsilonRI cross-linking. Our preliminary data suggest that Akt activity is regulated by conventional PKC isoforms (alpha, betal and betall) in mast cells. Based on these data, we hypothesize that C-terminal tyrosine phosphorylation of several PKC isoforms (a, betal, zeta, and lambda/I) by Syk recruits Grb-2/Sos complexes to activate Ras (Hypothesis 1) and that the conventional PKC isoforms phosphorylate the critical residue Ser-473 for Akt activation (Hypothesis 2). We also have preliminary data suggesting the role of C-terminal hydrophobic motif of PKCa in substrate recognition (Hypothesis 3). To characterize in depth the roles of PKC isoforms and Akt in mast cell activation, we will evaluate these hypotheses in in vitro and in vivo experiments. The proposed studies will bring novel insight into our understanding of mast cell signal transduction. Given the critical importance of PKC in degranulation and other activation events, these studies are also likely to provide an opportunity for novel therapeutic modalities aimed at allergic diseases.